一种用于检测1,2-苯醌和1,4-苯醌的白蛋白及血红蛋白加合物的新检测方法。

A new assay for albumin and hemoglobin adducts of 1,2- and 1,4-benzoquinones.

作者信息

Waidyanatha S, Yeowell-O'Connell K, Rappaport S M

机构信息

Department of Environmental Science and Engineering, School of Public Health, University of North Carolina at Chapel Hill 27599-7400, USA.

出版信息

Chem Biol Interact. 1998 Sep 4;115(2):117-39. doi: 10.1016/s0009-2797(98)00067-2.

DOI:10.1016/s0009-2797(98)00067-2

PMID:9826945

Abstract

A new method has been developed to detect mono-S-substituted cysteinyl adducts of 1,2- and 1,4-benzoquinone (BQ) in hemoglobin (Hb) and albumin (Alb). After reacting the protein with trifluoroacetic anhydride and methanesulfonic acid, the resulting isomers of O,O',S-tris-trifluoroacetyl-hydroquinone and -catechol are extracted and detected by gas-chromatography-mass spectrometry in the negative-ion chemical ionization mode. The limit of detection of the assay is about 20 pmol adduct/g protein. This assay was employed to quantitate mono-S-substituted background adducts in human and rat Hb and Alb and benzene-specific adducts in Hb and Alb from F344 rats following a single oral dosage of 50-400 mg [13C6]benzene/kg body wt. In Alb, a dose-related increase in both [13C6]1,2- and [13C6]1,4-BQ adducts was observed with [[13C6]]1,4-BQ-Alb] >> [[13C6]1,2-BQ-Alb]. The formation of [13C6]1,2-BQ-Alb was linear with increasing dosage of benzene with a slope of 2.3 (pmol adduct/g protein)/(mg/kg body wt.) (S.E. = 0.18, R2 = 0.91). However, at dosages above about 100 mg [13C6]benzene/kg body wt., the levels of 1,4-BQ-Alb were greater than proportional to the dosage. Mono-S-substituted adducts of [13C6]1,2-BQ and [13C6]1,4-BQ were not detected in Hb. The background ([12C6]) adducts of 1,2- and 1,4-BQ in 20 F344 rats were estimated (in nmol adduct/g of protein) to be 3.9 (S.E. = 0.23) and 4.9 (S.E. = 0.30) in Hb and 2.7 (S.E. = 0.24) and 11.4 (S.E. = 0.60) in Alb. At the highest dosage of 400 mg [13C6]benzene/kg body wt., background levels of 1,2-BQ-Alb were about 4-fold higher than those of the benzene-specific adducts whereas the benzene-specific levels of 1,4-BQ-Alb were about 7-fold higher than those of the background adducts. Background levels of 1,2- and 1,4-BQ adducts in 10 portions of commercial human proteins were found to be (in nmol adduct/g of protein) 1.6 (S.E. = 0.05) and 0.85 (S.E. = 0.04) in Hb and 1.6 (S.E. = 0.06) and 8.9 (S.E. = 0.36) in Alb.

摘要

已开发出一种新方法，用于检测血红蛋白（Hb）和白蛋白（Alb）中1,2 - 和1,4 - 苯醌（BQ）的单-S-取代半胱氨酸加合物。蛋白质与三氟乙酸酐和甲磺酸反应后，所得的O,O',S-三-三氟乙酰基-对苯二酚和-邻苯二酚异构体被萃取，并通过负离子化学电离模式的气相色谱-质谱法进行检测。该检测方法的检测限约为20 pmol加合物/克蛋白质。采用此检测方法对人和大鼠的Hb和Alb中的单-S-取代背景加合物以及F344大鼠单次口服剂量为50 - 400 mg [13C6]苯/千克体重后Hb和Alb中的苯特异性加合物进行定量。在Alb中，观察到[13C6]1,2 - 和[13C6]1,4 - BQ加合物均呈剂量相关增加，且[[13C6]]1,4 - BQ - Alb] >> [[13C6]1,2 - BQ - Alb]。[13C6]1,2 - BQ - Alb的形成与苯剂量增加呈线性关系，斜率为2.3（pmol加合物/克蛋白质）/（毫克/千克体重）（标准误差 = 0.18，R2 = 0.91）。然而，在剂量高于约100 mg [13C6]苯/千克体重时，1,4 - BQ - Alb的水平与剂量的比例关系更大。在Hb中未检测到[13C6]1,2 - BQ和[13C6]1,4 - BQ的单-S-取代加合物。估计20只F344大鼠Hb中1,2 - 和1,4 - BQ的背景（[12C6]）加合物（以nmol加合物/克蛋白质计）分别为3.9（标准误差 = 0.23）和4.9（标准误差 = 0.30），Alb中分别为2.7（标准误差 = 0.24）和11.4（标准误差 = 0.60）。在最高剂量400 mg [13C6]苯/千克体重时，1,2 - BQ - Alb的背景水平比苯特异性加合物高约4倍，而1,4 - BQ - Alb的苯特异性水平比背景加合物高约7倍。在10份市售人源蛋白质中，Hb中1,2 - 和1,4 - BQ加合物的背景水平（以nmol加合物/克蛋白质计）分别为1.6（标准误差 = 0.05）和0.85（标准误差 = 0.04），Alb中分别为1.6（标准误差 = 0.06）和8.9（标准误差 = 0.36）。